Cholesterol and hedgehog signaling

ABSTRACT

The present invention sterol-modified hedgehog polypeptides and functional fragments thereof Methods of identifying compositions which affect hedgehog activity based on inhibition of cholesterol modification of hedgehog protein are described. In one aspect of the invention, the method provides a means for affecting cholesterol biosynthesis or transport in a cell comprising contacting a cell with an effective amount of a compound that affects hedgehog, thereby affecting cholesterol biosynthesis or transport. The effect may be inhibition or stimulation of cholesterol biosynthesis or transport.

RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional applicationNo. 60/074,714, filed Feb. 13, 1998, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates generally to the field of proteinprocessing and protein signalling pathways and specifically to two novelproteins having distinct activities, which are derived from a commonhedgehog protein precursor.

[0004] 2. Related Art

[0005] Over the past decade, extracellular protein signals encoded byseveral gene families have emerged as central players in coordinatingcell behavior and thus generating pattern during animal development.Members of the hedgehog (hh) gene family in particular are notable fortheir association with several well-studied patterning activities. InDrosophila, where hh was discovered and isolated, patterning functionsinclude specification of positional identity within developing segmentsand appendages. In vertebrate embryos, function of the hh family memberSonic hedgehog (Shh) is associated with the patterning influences ofnotochord and prechordal plate mesoderm on spinal cord and brain, aswell as on other surrounding structures. Shh expressed in mesoderm atthe posterior margin of the developing vertebrate limb bud also plays acentral role in controlling limb outgrowth and patterning. Thepatterning functions of hh proteins have been extensively studied (seeHammerschmidt et al. 1997 for a recent general review), and novelfunctions continue to emerge.

SUMMARY OF THE INVENTION

[0006] This article presents a selective view of the hh proteinbiogenesis and signaling pathways, with particular attention paid to theinvolvement of the abundant neutral lipid cholesterol. One role forcholesterol is as a covalent adduct for the biologically active form ofthe hh protein (Hh), which is formed as a product of an autoprocessingreaction that entails internal cleavage. Cholesterol attachmentrestricts the spatial deployment of the Hh signal, thus influencing thepattern of cellular responses in developing tissues. Here we summarizeour studies of the Hh autoprocessing reaction, and of the role ofcholesterol in this reaction. We also summarize more recent studiessuggesting that, in addition to its role in Hh signal production,cholesterol has an essential role in mediating the response to the Hhsignal within target cells. This role is revealed by genetic ordrug-induced perturbations of cholesterol homeostasis that render targettissues unresponsive to the Hh signal.

[0007] In yet another embodiment, the invention provides a method foridentifying a compound which affects hedgehog activity comprisingincubating the compound with hedgehog polypeptide, or with biologicallyactive fragments thereof, or with a recombinant cell expressinghedgehog, under conditions sufficient to allow the components tointeract; and determining the effect of the compound on hedgehogactivity or expression. For example, cholesterol level (e.g.,biosynthesis or transport) is measured as an inidicator of hedgehogactivity. In one aspect of the invention, the method provides a meansfor affecting cholesterol biosynthesis or transport in a cell comprisingcontacting a cell with an effective amount of a compound that affectshedgehog, thereby affecting cholesterol biosynthesis or transport. Theeffect may be inhibition or stimulation of cholesterol biosynthesis ortransport.

DESCRIPTION OF THE FIGURES

[0008]FIG. 1. Autoprocessing of the Hedgehog protein precursor.Following signal sequence cleavage, the Hedgehog precursor (Hh)undergoes an autoprocessing reaction that entails cleavage between theGly and Cys residues within a tripeptide, Gly-Cys-Phe, that is conservedamong all Hh proteins; this cleavage is accompanied by attachment ofcholesterol to the carboxy-terminus of the amino-terminal product.Whereas the amino-terminal domain is active in signaling, thecarboxy-terminal domain mediates the autoprocessing reaction, and theresulting modification by cholesterol influences the tissue distributionof signaling acitivity. As indicated, amino acid sequence conservationamong orthologues of the Hh family is greater within the amino-terminalas compared to the carboxy-terminal domain. Crystallographic analysis ofthe amino-terminal domain of Shh protein revealed striking similarity infolded structure of a portion of this domain to the catalytic domain ofD,D carboxypeptidase, a zinc hydrolase from Streptomyces that acts oncell wall components (Dideberg et al. 1982; Hall et al. 1995; Murzin1996); the significance of this similarity and the role of this putativehydrolase in Shh signaling are not known. Not shown in this figure,N-linked glycosylation of carboxy-terminal sequences has been reportedwithin the carboxy-terminal domain of Shh (Bumcrot et al. 1995), but theglycosylation site is not uniformly conserved among Hh orthologues andits significance is unknown.

[0009]FIG. 2. Cell surface association of the amino-terminal domainIndirect immunofluorescence staining revealed a prominent cell-surfaceassociation of amino-terminal epitopes (A,B) with the plasma membrane;this staining was observed in the absence of detergent permeabilization,indicating localization to the cell surface The Western blot in (C)shows amino-terminal domain expression from full-length Hh protein(Hh-Np; lanes 1,2) or from a construct with a chain termination codon atthe cleavage site (Hh-N; lanes 3,4). Hh-Np is retained by cells withinthe culture whereas Hh-N is nearly quantitatively released into themedium. The greater mobility of Hh-Np relative to Hh-N is characteristicof processed Hh amino-terminal domains.

[0010]FIG. 3. Hh autoprocessing and protein self-splicing are initiatedby formation of a thioester intermediate. As described in the text, Hhautoprocessing (A) and protein self-splicing (B) are both inititated byformation of a thioester in place of a main-chain peptide. The reactionsdiffer in the second step, which for Hh entails attack of the thioesterintermediate by cholesterol. For self-splicing proteins, the secondnucleophile is the side chain of the first residue in thecarboxy-terminal extein (C extein); the resulting three-branchedintermediate is then resolved to give rise to the free intein andligated N and C exteins. For simplicity, proton transfers implicit inthe activation of nucleophiles or of leaving groups are omitted fromthis scheme.

[0011]FIG. 4. Evolutionary lineage of proteins containing Hint domains.Hint domains are found within three distinct protein families: theself-splicing proteins, the Hedgehog family of signaling proteins, and anovel family of C. elegans proteins of unknown function. In theself-splicing proteins an endonuclease domain is inserted within aperipheral loop of the Hint domain (Duan et al. 1997; Hall et al. 1997).In Hh proteins, a sterol recognition region (SRR) appended to thecarboxy-terminus of the Hint domain is required for cholesteroladdition; in the absence of SRR sequences, only the first step ofthioester formation occurs. The Hint domains of the C. elegans proteinsare more closely related to those of the Hh family, but SRR sequencesare replaced by other sequences of variable length, tentatively referredto as the adduct recognition region (ARR). Eleven C. elegans proteinsthat contain Hint domains have been identified thus far (at ˜80%completion of the C. elegans genomic sequence); this family can besubdivided further into two homologous groups based on the presence oftwo unrelated types of sequences present within the amino-terminaldomains of these proteins.

[0012]FIG. 5. Inhibition of cholesterol biosynthesis by the plantsteroidal alkaloid, jervine. Sterols were extracted and analyzed by HPLCfrom COS7 cells metabolically labelled with [3H]-mevalonic acid in thepresence or absence of jervine, a teratogenic plant steroidal alkaloid.In the presence of 28

M jervine, radiolabelled cholesterol levels were reduced and anotherradiolabelled sterol was found to accumulate. On the basis of itsretention time in this reverse phase HPLC method (Rodriguez and Parks1985), this abnormal sterol is tentatively identified as zymosterol, anintermediate in the cholesterol biosynthetic pathway

[0013]FIG. 6. Proteins with sterol sensing domains. Four proteinscontaining a sterol sensing domain (SSD) are schematically depicted. Thecylinders denote predicted transmembrane helices and the SSD of eachprotein is enclosed within the rectangle formed by the dashed lines. Asindicated by the shading, the homology between Patched (Ptc) and theNiemann-Pick C disease protein (NP-C) extends beyond the SSD to includeall twelve of the Ptc transmembrane domains. In the case of HMG CoAreductase, the topology of these transmembrane segments wasexperimentally determined (Olender and Simoni 1992; Roitelman et al.1992). The topology of Ptc shown is as suggested by Goodrich et al.(1996), and that of SCAP as suggested by Brown and Goldstein (1997). Theproposed topology of the NP-C protein is based upon sequence analysispresented by Carstea et al. (1997) and the homology to Ptc; not shownare several transmembrane domains that are weakly predicted to exist inthe human but not the mouse protein. The arrowhead after the firsttransmembrane domain of NP-C denotes a possible site of signal sequencecleavage as suggested by Carstea et al. (1997). The drawings onlycrudely approximate the extent of loops between transmembrane domainsand are not intended to convey structural information. The labellingwithin these loops indicates the presence of the catalytic domain incarboxy-terminal portions of HMG CoA reductase, of four repeats of theWD protein-protein interaction domain in the carboxy-terminal portion ofSCAP (Hua et al. 1996), and a region in the first loop of NP-C that istightly conserved among all NP-C homologues from various species(Carstea et al. 1997; Loftus et al. 1997).

DESCRIPTION OF THE INVENTION

[0014] Results and Discussion

[0015] Hedgehog Protein Autoprocessing

[0016]FIG. 1 presents a view of Hh biosynthesis which, although largelyderived from studies of Drosophila Hh, likely applies to Hh proteinsfrom all species. As suggested by genetic studies (Mohler 1988) and aspredicted from sequence analysis (Lee et al. 1992; Mohler and Vani 1992;Tabata et al. 1992; Tashiro et al. 1993), the Hh protein enters thesecretory pathway and is cleaved following a signal sequence locatednear the amino terminus (Lee et al. 1992). From earliest examination inin vitro translation experiments, the Drosophila Hh protein alsorevealed a propensity to undergo cleavage at another internal site (Leeet al. 1992); antibodies specifically directed against amino- orcarboxy-terminal epitopes confirmed that the predominant forms ofendogenous Hh protein correspond to the products of this internalcleavage (Lee et al. 1994). The internal cleavage depends uponcarboxy-terminal Hh sequences and can be observed with purifiedrecombinant protein in vitro, thus indicating the operation of aself-directed processing activity (Lee et al. 1994).

[0017] Further in vitro analysis of this cleavage demonstrated that itoccurs between the Gly and Cys residues within a conserved Gly-Cys-Phetripeptide (Porter et al. 1995). This information permitted ectopicexpression in transgenic Drosophila of constructs encoding either theamino-terminal or the carboxy-terminal cleavage products (Hh-N and Hh-C,respectively), and these studies demonstrated that biological signalingactivity resides entirely within the precisely truncated Hh-N fragment(Porter et al. 1995). Similar transgenic experiments also demonstratedthat mutations within Hh-C that interfere with processing but do notalter Hh-N sequences nevertheless block Hh function (Lee et al. 1994;Porter et al. 1995). Thus, whereas Hh-N suffices for signaling activity,Hh-C sequences are required to generate the active amino-terminalsignaling domain from precursor via autoprocessing (FIG. 1). Consistentwith these conclusions, all molecularly characterized hh mutations inDrosophila either directly affect the Hh-N signaling domain or otherwiseappear to block release of the signaling domain from precursor byaffecting the Hh-C autoprocessing function (Porter et al. 1995).

[0018] Biological Role of Autoprocessing

[0019] Since the cleavage products of the Hh precursor are thepredominant forms observed in vivo, the occurrence of the autoprocessingevent appears not to be regulated. What then is the raison d'etre ofautoprocessing? The answer to this question began to emerge from studiesin cultured cells which demonstrated that processed amino-terminaldomain protein generated from precursor remains tightly associated withthe cell surface (Lee et al. 1994; FIG. 2a,b). In contrast, proteinexpressed from a construct lacking the autoprocessing domain is almostquantitatively released into the culture medium (Porter et al. 1995;FIG. 2c). Autoprocessing thus is associated with tethering of theamino-terminal signaling domain to the cell surface. Amino-terminaldomain protein derived by processing of the Hh precursor is designatedHh-Np, to distinguish it from amino-terminal domain derived from atruncated construct (Hh-N).

[0020] As would be expected for a potent secreted signal whoseexpression is spatially restricted within segments, autoprocessing andcell surface tethering play an important role in segmental patterning(Porter et al. 1996 a). This role was revealed by comparing the effectsof localized expression of Hh-N or Hh-Np in transgenic Drosophilaembryos: when activated at the normal sites of hh transcription,transgenes expressing Hh-Np, which is also the form of the endogenousprotein signal, showed no significant alterations in the normalsegmental patterns of gene expression or cuticle formation. In contrast,similar localized expression of transgenic Hh-N caused disruption ofsegmental patterning equivalent to that caused by ubiquitous high-levelexpression of Hh protein. Autoprocessing thus appears to restrict thespatial distribution of Hh signaling activity, and immunofluorescencestudies indeed confirm that Hh-N more readily diffuses from expressingcells to surrounding cells than does Hh-Np (Porter et al. 1996a). Theseimmunofluorescence studies also revealed a difference in subcellularlocalization within expressing cells, with Hh-Np sequestered in largepunctate structures in basolateral domains of epidermal cells. Hh-Nprotein from the truncated construct in contrast lacks this type ofpunctate localization and instead appears to be freely secreted to theapical surface of expressing cells within the epidermal epithelium(Porter et al. 1996a). The functional significance of thisprocessing-dependent localization to the basolateral domain ofexpressing cells within the epidermal epithelium is not yet known.

[0021] The biological role of autoprocessing in vertebrates isparticularly well illustrated by the role of Shh in neural tubepatterning. Shh protein is processed similarly to the Hh protein(Bumcrot et al. 1995; Chang et al. 1994; Ekker et al. 1995; Lai et al.1995; Porter et al. 1995; Roelink et al. 1995), and the normal cellsurface association of the amino-terminal fragment also isprocessing-dependent (Bumcrot et al. 1995; Porter et al. 1996b; Roelinket al. 1995). Loss of Shh gene function in mouse embryos results infailure to differentiate floor plate cells and motor neurons (Chiang etal. 1996). Induction in naive neural plate explants of these and otherventral cell types by recombinant Shh-N protein occurs in aconcentration-dependent manner, with motor neurons induced at lowconcentrations and floor plate cells induced at the expense of motorneuron fates at higher concentrations (Ericson et al. 1997; Marti et al.1995; Roelink et al. 1995). In other explant experiments with embryonictissues as inducers, structures such as the notochord can only inducefloor plate cells in a contact-dependent manner (Placzek et al. 1993)whereas motor neuron induction does not require such contact (Yamada etal. 1993). The ability to circumvent contact-dependence with highconcentrations of soluble Shh-N protein suggests that one role formodification and surface association of the signaling domain is togenerate large concentration differences between local and distantsites, with consequent sharp distinctions between the cell typesinduced. Consistent with this idea, the Shh signaling domain is foundpredominantly on the surface of notochord cells and embryonic floorplate normally forms only in close proximity to the notochord.

[0022] The Autoprocessing Reaction

[0023] Given the striking differences in diffusibility and patterningactivity of Hh-N and Hh-Np, it was not surprising to find accompanyingphysical differences. As compared to Hh-N, Hh-Np displays a slightdifference in electrophoretic mobility (FIG. 2c), a dramatic increase inhydrophobic character, a greater mass associated with thecarboxy-terminal fragment of CNBr digestion, and an insensitivity todigestion by carboxypeptidase (Porter et al. 1996a). These data togetherindicate that Hh-Np carries a covalently attached lipophilic adduct atits carboxy-terminus whose addition depends upon the autoprocessingactivity of the carboxy-terminal domain. The presence of this adductaccounts for the tethering of Hh-Np to the cell surface, since the lipidadduct would be expected to partition preferentially into the lipidbilayer.

[0024] Despite information about its mass and other properties, theidentity of the lipid adduct could not be determined directly becausequantities of purified Hh-Np sufficient for chemical analysis proveddifficult to obtain. Identification of the adduct therefore reliedultimately on a mechanistic understanding of the in vitro processingreaction and its use as an assay to identify a lipid capable ofparticipating in the autoprocessing reaction. Initial insight into theautoprocessing reaction derived from the observation that the kineticsof cleavage in vitro were independent of starting protein concentration,indicating an intramolecular mechanism (Porter et al. 1995). From alimited number of proteins known to autoprocess by an intramolecularmechanism, a particularly strong analogy could be drawn to prohistidinedecarboxylase (van Poelje and Snell 1990), which is capable ofundergoing intramolecular cleavage with either a Cys or Ser residue atthe position immediately following the scissile bond; Hh autocleavagealso could be observed in vitro, albeit inefficiently, if a Ser residuereplaced the normal Cys (Porter et al. 1996a). Contemporaneously withour studies of Hh autoprocessing, the self-splicing proteins have alsoemerged as intramolecular processing proteins with Ser or Cys residuesat the site of cleavage (Xu and Perler 1996); we now know that thesimilarities between Hh and self-splicing proteins extend beyondmechanism to include sequence and structure (Hall et al. 1997; seebelow).

[0025] The feature common to all of these autoprocessing reactions isinitiation by attack of a nucleophilic side chain upon the precedingcarbonyl, with displacement of the peptide amine and formation of anester or thioester intermediate. As seen in FIG. 3A, this is the firststep of the Hh autoprocessing reaction, with a labile thioesterreplacing the main chain peptide bond between amino- andcarboxy-terminal domains (Porter et al. 1996a; Porter et al. 1995). Thesecond step of the Hh autoprocessing reaction involves attack upon thesame carbonyl by a second nucleophile, displacing the sulfur andsevering the connection between Hh-N and Hh-C. The requirement for asecond nucleophile in vitro can be met by a high concentration either ofa thiol-containing molecule or of another small molecule withnucleophilic properties at neutral pH; these small nucleophiles can beshown to form covalent adducts to the amino-terminal product of the invitro cleavage reaction (Porter et al. 1996a).

[0026] Of some interest in the in vitro studies of Hh autoprocessing wasthe use of Cys-initiated peptides as nucleophile in the second step: theinitial linkage between the peptide and the amino-terminal product is athioester, which can then rearrange to form an amide bond by reversal ofthe steps involved in thioester formation during the first part of thereaction (Porter et al. 1996a). The net effect of these reactions is theligation of a Cys-initiated peptide at the site of cleavage, and isanalogous to the recent use of a chemically synthesized thioesterintermediate for peptide ligation (Dawson et al. 1994).

[0027] Cholesterol Modification In Vitro and In Vivo

[0028] To account for the lipid modification in Hh-Np, the in vivoreaction was presumed to occur with the participation of an endogenouslipid carrying the second nucleophilic moiety. This presumption led touse of the in vitro reaction as an assay which was applied tofractionated cell lipids, leading to the identification of cholesterolas a neutral lipid that at relatively low concentrations could supplythe requirement for a nucleophile in the second step (Porter et al.1996b). Cholesterol thus stimulates the in vitro autoprocessing reactionand forms a covalent linkage to the amino-terminal product of cleavagereaction. This linkage is sensitive to base treatment, consistent withformation of an ester with the oxygen of the 3° hydroxyl of cholesterol.Confirming this role for cholesterol in vivo, [3H]-cholesterol wasobserved to label Hh-Np or Shh-Np expressed in Drosophila or inmammalian cultured cells, and this label could be removed by basetreatment (Porter et al. 1996b; K.E.Y, J.A.P. and P.A.B., unpublishedresults). The label hydrolyzed from Hh-Np was further analyzed and shownto display chromatographic behavior identical to that of cholesterol,indicating that the in vivo adduct is cholesterol and not some othersterol derivative.

[0029] A somewhat surprising finding in the metabolic labellingexperiments with mammalian cells is the apparent linkage of cholesterolto several other mammalian proteins. There is little evidence at presentregarding the identity and function of these proteins, or the mechanismof attachment of cholesterol. We have found that the cholesterol can beremoved from these proteins by base treatment, suggestive of an esterlinkage like that resulting from Hh autoprocessing (K.E.Y, J.A.P andP.A.B., unpublished results).

[0030] Thioesters as Intermediates in Protein Modification

[0031] The use of a Cys-derived thioester as an intermediate is a themecommon to several other acyl transfers that result in covalentmodifications of proteins. Following formation of the initial thioesterin these systems, the acyl portion of the thioester (the acceptor,corresponding to Hh-N; see diagram in Table 1) can receive the finalmodification directly or alternatively may be transferred to otherthiols in one or more subsequent steps before receiving the finalmodification is (Table 1). The ubiquitin cascade represents such areaction with multiple intermediates, whose role is to attach ubiquitinto proteins destined for degradation by the proteasome (Hochstrasser1996). The acyl group for these thioesters is supplied by thecarboxy-terminal Gly of ubiquitin, and the thiols come from Cys sidechains in three distinct classes of enzymes. The first of these, E1,forms the initial thioester in an ATP-consuming reaction. Then, throughtrans(thio)esterification reactions, the ubiquitin forms thioesterssequentially with E2 and E3 enzymes, before final transfer to the eamine of a Lys side chain. The protein receiving ubiquitin in theresulting amide linkage is thus marked for degradation.

[0032] The a-macroglobulin proteinase inhibitors and the C3, C4, and C5complement proteins represent members of an ancient superfamily that usean intrachain thioester as a “spring loaded” functionality that can betriggered for covalent attachment to target molecules (Chu and Pizzo1994). The intrachain thioester is formed by thiol attack of a Cys sidechain on the amido group of a Gln side chain. The final adducts in thecase of the complement proteins are nucleophiles on the surface of cellsto be targeted for lysis. In the a-macroglobulin case, the final adductis a nucleophile on a protease to be inactivated, which is targeted toa-macroglobulin through the presence of multiple cleavage sites forproteases of various specificities.

[0033] In the examples just discussed, the acyl group contributing tothe thioester intermediate derives either from another protein or froman amino acid side chain. In contrast, the acyl group in the Hhthioester intermediate is linked to a main chain carbonyl, and thethioester therefore replaces an amide bond within the peptide backbone.Other proteins likely to utilize main chain ester or thioesterintermediates in autoprocessing reactions include prohistidinedecarboxylase and certain members of the Ntn hydrolase family that areprocessed by an intramolecular mechanism (Brannigan et al. 1995; Guan etal. 1996). The Ntn (N-terminal nucleophile) hydrolases are structurallyrelated enzymes that are autoprocessed with internal cleavage, leavingthe active site nucleophile as the amino-terminal residue. The role ofthese reactions appears to be activation of a precursor protein andthere is no net addition of a modifying adduct. Thus, although theprohistidine decarboxylase reaction was of heuristic value inunderstanding the mechanism of Hh autoprocessing, there is no evidenceof any evolutionary relationship between Hh autoprocessing domains andeither prohistidine decarboxylase or Ntn hydrolase proteins.

[0034] Ester Intermediates in Proteins Containing the Hint Domain

[0035] In contrast, Hh proteins are evolutionarily related to two othergroups of proteins, the self-splicing proteins and a group of novelnematode proteins containing Hh-C-like sequences. The self-splicingproteins undergo a reaction in which an internal portion of the protein,termed an intein, is excised and amino- and carboxy-terminal flankingregions, termed exteins, are ligated to form the mature protein (Perleret al. 1994). Inteins are found inserted into a wide variety ofarchaeal, bacterial, chloroplast, and yeast proteins. The intein portionmediates the protein splicing reaction and typically also contains anendonuclease thought to act at the DNA level in mediating movement ofintein coding sequences. Similar to Hh autoprocessing, the proteinsplicing reaction is initiated by intramolecular attack of a hydroxyl orthiol upon the preceding carbonyl, and the resulting ester or thioesterintermediate replaces the peptide bond at the amino-terminalextein/intein boundary (Xu and Perler 1996); FIG. 3). Unlike Hhproteins, the second nucleophilic attack in the protein self-splicingreaction involves the side chain of another Ser or Cys residue severalhundred residues downstream. The resulting branched protein intermediateultimately resolves into the ligated exteins and the free intein protein(FIG. 3).

[0036] Nematode proteins with Hh-C-like sequences were identified bysearching for homology within the C. elegans genomic sequence database.At ˜80% completion of the C. elegans genome, eleven putative proteinswith homology to the Hh-C autoprocessing domain have been identified(Burglin 1996; Hall et al. 1997; Porter et al. 1996a; R. Mann, X. Wang,and P.A.B., unpublished data). As in the Hh family, the Hh-C-like domainis located at the carboxy-terminus of these proteins and is preceded byan amino-terminal domain bearing a signal sequence. The amino-terminaldomains of these nematode proteins, however, bear no sequence similarityto Hh-N. Instead, they resemble each other and can be divided into twofamilies. The structures of these proteins suggest the possibility thatthey are secreted and undergo autoprocessing; a preliminary study of onefamily member in Drosophila cultured cells indeed demonstrates cleavageat the junction between amino- and carboxy-terminal domains (Porter etal. 1996a).

[0037] The level of amino acid sequence identity between these nematodeproteins and Hh ranges from 24 to 32% in a region approximatelycorresponding to the amino-terminal ⅔ of Hh-C. This same region of Hh-Calso can be aligned with inteins, although alignment is complicated bythe presence of sequences corresponding to the endonuclease (Dalgaard etal. 1997; Hall et al. 1997; Pietrokovski 1997). The level of amino acididentity between Hh-C and inteins with endonuclease sequences removed is˜10%, but most of the residues known to be essential for Hh-C processingactivity are conserved.

[0038] A common evolutionary origin for these protein families isfurther indicated by a domain with a common fold that is present in thecrystal structure of a portion of Hh-C and in the crystal structure ofthe 454 residue intein protein PI-SceI (Duan et al. 1997; Hall et al.1997). Two additional domains not present in the Hh-C fragment arepresent in the intein structure: one of these is the endonuclease andthe other is thought to aid in DNA binding. Remarkably, both of theseadditional domains are inserted into peripheral loops of the commondomain, with little apparent effect upon its three dimensional fold. Thecrystallized Hh fragment contains the amino-terminal 151 residues ofHh-C, of which the first 145 residues are well-ordered in the crystalstructure; these residues correspond to the region conserved in thenematode proteins. This domain alone suffices for thioester formation,as indicated by the ability of a Hh protein truncated after this pointto undergo cleavage in the presence of DTT (Hall et al. 1997), and thisdomain has been referred to as the Hint module (Hedgehog, intein).

[0039] Although the Hint module in Hh-C suffices for the first step ofautoprocessing, at least some part of the 63 carboxy-terminal residuesmissing in the crystallized fragment are required for the second step ofcholesterol addition (Hall et al. 1997). Because of its apparent role insterol addition, this 63 residue region d us to be acutely aware of theis referred to as SRR, for sterol recognition region. No clear alignmentcan be made between SRR and sequences within the nematode family,however, and sequences in these nematode proteins that extendcarboxy-terminal to the Hint domain are tentatively designated ARR, foradduct recognition region. The differences in sequence between the SRRof Hh proteins and the ARR regions of nematode gene family membersraises the possibility that molecules other than cholesterol mayparticipate in the processing reaction and form novel protein-modifyingadducts.

[0040] From these sequence and structure relationships, a plausibleevolutionary history can be constructed in which all three proteingroups diverged from an ancestral Hint domain (Hall et al. 1997; seeFIG. 4). In one branch, the ancestral intein was formed by insertion ofan endonuclease into a Hint domain and by adjustment (or preservation)of the chemistry to insure that the second nucleophilic attack is madeintramolecularly by the side chain of a downstream residue. In a secondbranch, Hh proteins were formed by association of a Hint domain withamino-terminal domains of the Hh and nematode proteins. The sequence ofevents leading to formation of these proteins is not known. Onepossibility is that the Hint and SRR modules may have been assembledinto a cholesterol transfer unit prior to association with the Hhsignaling domain; alternatively, the Hint module might have beeninserted within a preassembled protein comprising a signaling domain andthe SRR precursor. In the second scenario, the SRR precursor in thepreassembled protein might have served some function related to sterolrecognition, such as membrane association. Similarly, several scenariosare possible in assembly of the nematode proteins. The possibility alsoexists that additional proteins will be found in which the Hint moduleinitiates novel splicing or transfer reactions.

[0041] Cholesterol Synthesis Inhibitors and Holoprosencephaly

[0042] One of the most striking aspects of the Shh loss-of-functionphenotype in mice (Chiang et al. 1996) is its resemblance toholoprosencephaly (HPE), a term applied to a spectrum of humandevelopmental malformations characterized by a loss of midlinestructures in the forebrain and face. In its most severe form, as seenin Shh −/− mice, HPE is associated with a cyclopic eye positionedbeneath a proboscis consisting of fused nasal chambers (Cohen and Sulik1992). Abnormal features of brain anatomy, for which the syndrome isnamed, include an absence of ventral forebrain structures anddevelopment of remaining forebrain structures as a single fused vesicle.Experimental manipulations of amphibian embryos carried out more than 60years ago led to an understanding of cyclopia as a consequence ofdisrupting the influence normally exerted by prechordal plate mesodermupon forebrain neuroepithelium (Adelmann 1936b; Adelmann 1936a; Mangold1931). This influence is required for bilateral subdivision of the earlyeye field; in its absence the eye field remains continuous across themidline, resulting in cyclopia and the loss of such ventral forebrainderivatives as the pituitary and the optic chiasm.

[0043] Shh expression in the prechordal mesoderm underlying the neuralplate can first be detected in mid-streak mouse embryos (Chang et al.1994; Echelard et al. 1993), a stage that coincides with or justprecedes the requirement for prechordal plate signaling. All of thesestudies therefore are consistent with the view that Shh constitutes orcontributes to the midline signal that passes from the prechordal platemesoderm to forebrain neural plate, in a manner analogous to that inwhich Shh from the notochord induces regionalization and morphogenesisof the spinal cord. Recent studies indeed have demonstrated that anautosomal dominant form of human HPE is caused by mutations in the humanShh gene (Belloni et al. 1996; Roessler et al. 1996). The mutationsdescribed would be expected to cause a loss of Shh function, indicatingthat in contrast to the mouse Shh mutation, which is entirely recessive(Chiang et al. 1996), human Shh function is haploinsufficient.Consistent with this interpretation, the malformations associated withheterozygous human Shh mutations are variable, even among individualscarrying the same allele, and are far less severe than those in thehomozygous mouse Shh mutation (Chiang et al. 1996).

[0044] Given this association of HPE with mutations in the Shh gene, areasonable supposition would be that HPE could also be caused by otherperturbations of the Shh signaling pathway. Of particular interest tous, in view of the role of cholesterol in Hh autoprocessing, were aseries of observations published beginning more than thirty years agowhich noted that HPE-like malformations can be induced by treatment ofpregnant rats with the drugs Triparanol, AY 9944R, and BM 15.766 (Dehartet al. 1997; Roux 1966; Roux 1964; Roux et al. 1979). These drugsinhibit enzymes of cholesterol biosynthesis and cause an abnormalaccumulation of desmosterol (Triparanol) or of 7-dehydrocholesterol (AY9944R and BM 15.766), which are the immediate precursors in alternatebiosynthetic routes to cholesterol.

[0045] Genetic Perturbations of Cholesterol Synthesis and Transport

[0046] Further links between cholesterol and vertebrate embryonicdevelopment are provided by several mouse and human mutations affectingcholesterol synthesis or transport. Smith-Lemli-Opitz Syndrome (SLOS) isan autosomal recessive human genetic disease characterized by numerousdevelopmental defects including microcephaly, pituitary agenesis, limband genital abnormalities, and defects of the heart, kidneys, andpancreas (Opitz 1994; Salen et al. 1996; Tint et al. 1994). Thesepatients lack the activity of 7-dehydrocholesterol reductase, the sameenzyme inhibited by AY 9944R or BM 15.766, and as a consequence haveabnormally low serum cholesterol levels and accumulate7-dehydrocholesterol. Approximately 5% of SLOS patients display signs ofHPE, with malformations that tend toward the milder end of the spectrum(Kelley et al. 1996). A possible explanation for the reduced severity ofthe defects as compared to those in the progeny of drug-treated rats ischolesterol supplementation from heterozygous mothers via placentalexchange. Consistent with this idea, high dietary cholesterol cansuppress the teratogenic effects of cholesterol synthesis inhibitorsgiven to pregnant rats (Roux et al. 1979). Some of the developmentalmalformations in SLOS patients are likely to result from deficiencies insteroid hormone biosynthesis as well as from effects on other unknowntargets.

[0047] Related developmental defects have also been described in mousemutants lacking function of the endocytic receptor megalin orapolipoprotein B (Herz et al. 1997). The megalin protein, also referredto as gp330, is encoded by a member of the low density lipoprotein (LDL)receptor gene family and is specifically expressed on the apicalsurfaces of embryonic neuroectoderm and neuroepithelium in thedeveloping neural tube. Apolipoprotein B (apoB) is the major structuralcomponent of several lipoprotein particles that carry esterifiedcholesterol and other neutral lipids in the circulation. The defects inmegalin-deficient mice include fusion of forebrain structures into asingle vesicle, agenesis of the olfactory bulbs and pituitary, andabsence of the corpus callosum, all malformations within theholoprosencephaly sequence and therefore suggestive of a perturbation inthe Shh signaling pathway (Willnow et al. 1996). The defects in micelacking apoB function are more severe, appear less specific and causeresorption of most homozygous embryos by 9.5 or 10.5 days of gestation(Farese et al. 1995; Huang et al. 1995). Mutations in both of thesegenes affect cholesterol transport, with the difference that the megalineffect may be restricted to cholesterol uptake in neural precursorswhereas the apoB defect would block all embryonic absorption ofmaternally derived cholesterol, normally transported via the yolk sac inthe mouse (Farese et al. 1996). A hypomorphic mutation that produces atruncated but functional apoB protein is homozygous viable and does notconsistently show developmental defects (Homanics et al. 1993). Thereduced cholesterol levels in these mice, however, makes themsusceptible to teratogenesis with the compound BM 15.766 and theresulting defects, including holoprosencephaly, are like those intreated rats (Lanoue et al. 1997). Normal mice are not susceptible totreatments with cholesterol synthesis inhibitors, possibly because theircholesterol levels are higher than those in rats.

[0048] Plant Teratogens as Cholesterol Synthesis Inhibitors

[0049] Another experimental model for holoprosencephaly derives from theoccurrence of epidemics of congenital craniofacial malformations amongnewborn lambs on sheep ranches in several National Forests of thewestern United States (Gaffield and Keeler 1996). The most dramaticallyaffected lambs showed severe holoprosencephaly, including true cyclopiaand other craniofacial malformations characteristic ofholoprosencephaly. The occurrence of these defects was traced to grazingby pregnant ewes on the range plant Veratrum californicum (Binns et al.1963). The compounds responsible were identified by Keeler and Binns(1968) as a family of steroidal alkaloids; the structures of two ofthese, cyclopamine and jervine, are shown as compared to cholesterol inFIG. 5A.

[0050] Given the structural similarities of these compounds tocholesterol and the similar teratogenic effects of cholesterol synthesisinhibitors upon the offspring of pregnant rats, a reasonable mechanismto consider for the effects of these plant sterol derivatives was theinhibition of cholesterol biosynthesis. Accordingly, we tested COS7cultured cells treated with jervine for defects in cholesterolbiosynthesis by labelling with [3H]-mevalonic acid and then extractingand analyzing radiolabelled, non-saponifiable lipids. FIG. 5B shows thattreated cells synthesized reduced levels of cholesterol and accumulatedincreased levels of another sterol that we have provisionally identifiedas the cholesterol precursor, zymosterol. The natural product jervine atthese concentrations thus inhibits cholesterol biosynthesis in culturedcells in much the same manner as the synthetic drugs discussed above,although the specific enzyme(s) affected appear to differ. Given thesimilarities in their teratogenic effects, this inhibition seems likelyto underlie the teratogenic effects of both the synthetic and naturalcompounds

[0051] Perturbations of Cholesterol Homeostasis Block the Response toShh Signaling

[0052] As reviewed above, there is a striking correspondence between thedevelopmental malformations in mouse and human Shh mutants and thosecaused by perturbations of cholesterol homeostasis These malformationsare caused by effects on either synthesis or transport of cholesterol;in the case of the hypomorphic apob allele combined with BM 15.766treatment, effects on both synthesis and transport appear to synergizein generating severe holoprosencephaly in mice, where neither effectalone suffices (Lanoue et al. 1997) The developmental malformationscaused by these perturbations of cholesterol homeostasis stronglysuggest that the Shh signaling pathway or its targets must somehow beaffected. Our attention initially was drawn to these perturbationsbecause of the role of cholesterol in Hh autoprocessing and thepossibility that autoprocessing and hence signal production might beaffected. But a second possibility is that instead of an effect onsignal production these perturbations of cholesterol homeostasis mightinterfere with the ability of target tissues to sense or transduce theShh signal. To distinguish these two possibilities we have examined theeffects of these synthetic and plant-derived compounds on theautoprocessing reaction in vivo and in vitro, and have also tested theability of drug-treated neural plate explants to respond to recombinantShb protein.

[0053] The autoprocessing reaction is not inhibited by these compoundsin cultured cells, nor is cleavage and cholesterol modificationinhibited in the in vitro reaction (M.K.C., J.A.P., K.E.Y, P.A.B.,manuscript in preparation). The amino-terminal product of processing indrug-treated cultured cells displays a mobility suggestive of sterolmodification. Since a number of cholesterol biosynthetic precursors areable to participate in the in vitro reaction, the adduct could either becholesterol or one of the precursors whose accumulation is caused bydrug treatment. The lack of any apparent effect on processing leavesopen the second possibility, that drug treatment affects the ability oftarget tissues to respond to the Shh signal. This possibility issupported by our observations with neural plate explants, in whichtarget genes normally induced by recombinant Shh-N protein areunresponsive when the explants are treated with synthetic andplant-derived cholesterol synthesis inhibitors (M.K.C., J.A.P., K.E.Y,P.A.B., manuscript in preparation). An effect on target tissues also isconsistent with the occurrence of HPE in megalin mutant mice and withthe specific expression of megalin in embryonic neuroepithelium duringthe critical period of Shh signaling (Willnow et al. 1996). The megalinreceptor is able to mediate LDL uptake (Stefansson et al. 1995), and nodevelopmental defects are observed in mice or humans lacking function ofthe more generally expressed LDL receptor (Ishibashi et al. 1993).Thissuggests that megalin may function specifically to maintain cholesterolhomeostasis in developing neuroepithelium, which is the target of Shhprotein signaling during the HPE is critical period. A link betweencholesterol homeostasis and activity of the Hh pathway is interestingsince most responses to Hh signaling in neuroepithelium or otherdeveloping tissues involve cell proliferation. This linkage might makesense from a regulatory point of view, given the importance ofcholesterol as a membrane component in dividing cells.

[0054] Additional information consistent with a role for cholesterol inreceiving the Shh signal derives from the recent isolation of theNiemann-Pick C (NP-C) disease gene from mouse and human (Carstea et al.1997; Loftus et al. 1997). The NP-C protein contains 13-16 transmembranedomains, and two features of its sequence are notable in the context ofShh signaling (Carstea et al. 1997; Loftus et al. 1997). The first isthat it resembles that of the Hh pathway protein Patched (Ptc)throughout most of its extent, and the second is that five of the NP-Ctransmembrane domains constitute an apparent sterol sensing domain(SSD), which had not previously been noticed in Ptc.

[0055] SSD sequences are also present in HMG-CoA reductase and SCAP(SREBP cleavage activating protein; Hua et al. 1996), two proteinsinvolved in maintenance of cholesterol homeostasis (FIG. 6). Although itis not yet known whether the SSD binds cholesterol directly or insteadindirectly senses cholesterol-induced changes in membrane properties(see e.g., Nezil and Bloom 1992), the SSD in these proteins is requiredfor a response to distinct levels of cellular cholesterol. In the caseof HMG-CoA reductase, the rate-limiting enzyme in the cholesterolbiosynthetic pathway, the SSD mediates decreased enzyme stability underconditions of cholesterol excess (Gil et al. 1985). The SCAP proteinresponds to sterol levels by modulating the cleavage and activation ofSREBP (sterol response element binding protein), a transcription factorthat controls expression of proteins involved in cholesterol synthesisand uptake (Brown and Goldstein 1997); in the absence of cleavage SREBPremains anchored to the endoplasmic reticulum via two transmembranedomains. The NP-C protein itself appears to play a role in cholesterolhomeostasis by directly acting in or by regulating cholesteroltransport, as indicated in NP-C cultured cells by a delayed response tochallenge by LDL with a consequent accumulation of cholesterol inlysosomes.

[0056] The Ptc protein has been suggested to constitute or contribute tothe Hh receptor mechanism, but its role is not that of a conventionalreceptor. In the absence of Ptc function the Hh signaling pathway isconstitutively activated (Ingham et al. 1991) The normal function of Ptcthus seems to be suppression of the pathway in target cells, and thissuppression is alleviated by the presence of the Hh signal. In additionto this cell autonomous role in suppressing the pathway, the Ptc proteinhas a role in sequestration of the Hh signal within tissues, a non-cellautonomous activity which affects the spatial extent of Hh signaling(Chen and Struhl 1996). These two activities can be geneticallyuncoupled, as demonstrated by a mutant protein that retains thesequestration function but, like other Ptc mutations, does not suppressthe Hh pathway (Chen and Struhl 1996). Recent biochemical evidencesuggests that Shh-N protein may interact directly with the mouse Ptcprotein, (Marigo et al. 1996; Stone et al. 1996) but, assuming that adirect interaction can be confirmed with the use of purified components,it is unclear whether it would play a role in either, both, or neitherof the Ptc functions described above.

[0057] These biochemical experiments were carried out with recombinantprotein that lacked the cholesterol modification (Marigo et al. 1996;Stone et al. 1996). In light of the presence of an SSD within Ptc, itwould be interesting to know whether the Hh cholesterol adductinfluences the apparent affinity of the amino-terminal signaling domainfor Ptc. Along these lines it is interesting to note that Drosophilaembryos expressing a truncated hh construct in the normal spatialpattern (Porter et al. 1996a) are similar to embryos that ectopicallyexpress full-length Hh and to embryos that lack Ptc function (Chang etal. 1994; Ingham 1993; Porter et al. 1996a): all three of thesegenotypes show spatially indiscriminate activation of the Hh pathway.One possible explanation of these similarities is that the cholesteroladduct not only increases the association of Hh signaling domain withproducing cells but also contributes favorably to the interaction withPtc. In the absence of the cholesterol adduct, the Hh-N protein wouldnot be as effectively sequestered by Ptc, leading to ectopic Hh pathwayactivation.

[0058] An enhanced sequestration by Ptc of the cholesterol-modifiedsignaling domain would represent a role distinct from that of mediatingthe response to cholesterol homeostasis within the target cell, althoughit is conceivable that the Ptc SSD might be involved in both. Given ourincomplete knowledge of the mechanistic role of Ptc, all discussion asto the role of its SSD must be considered speculative. But the presenceof a SSD in a protein with such a central role in regulating the Hhresponse is tantalizing as the possible link to the dual roles ofcholesterol in limiting the spatial extent of Hh signaling and infacilitating transduction of the Hh signal within target cells.

[0059] An experimental model for holoprosencephaly derives from theoccurrence of epidemics of congenital craniofacial malformations amongnewborn lambs on sheep ranches in several National Forests of thewestern United States. The most dramatically affected lambs showedsevere holoprosencephaly, including true cyclopia and other craniofacialmalformations characteristic of holoprosencephaly. The occurrence ofthese defects was traced to grazing by pregnant ewes on the range plantVeratrum californicum. The compounds responsible were identified as afamily of steroidal alkaloids; the structures of two of these,cyclopamine and jervine, are shown as compared to cholesterol in FIG.33. In FIG. 33, sterols were extracted and analyzed by HPLC from COS7cells metabolically labelled with [³H]-mevalonic acid in the presence orabsence of jervine, a teratogenic plant steroidal alkaloid. In thepresence of 28 mM jervine, radiolabelled cholesterol levels were reducedand another radiolabelled sterol was found to accumulate. On the basisof its retention time in this reverse phase HPLC method, this abnormalsterol is tentatively identified as zymosterol, an intermediate in thecholesterol biosynthetic pathway.

[0060] Given the structural similarities of these compounds tocholesterol and the similar teratogenic effects of cholesterol synthesisinhibitors upon the offspring of pregnant rats, a reasonable mechanismto consider for the effects of these plant sterol derivatives was theinhibition of cholesterol biosynthesis. Accordingly, COS7 cultured cellstreated with jervine were tested for defects in cholesterol biosynthesisby labelling with [3H]-mevalonic acid and then extracting and analyzingradiolabelled, non-saponifiable lipids.

[0061] Metabolic labeling and sterol analysis was essentially asdescribed (Popjak et al. J. Biol. Chem. 264: 630-6238.1989; Rilling etal. 1993 Arch. Biochem. Biophys. 301: 210-215.), with minormodifications. Briefly, COS-7 cells were plated at ˜35% confluence intotwo 60 mm dishes at 37° C. in 4 ml each of Dulbecco's modified Eagle'smedium (DMEM) supplemented with 10% fetal bovine serum (FBS). After 24hr of growth the medium in each dish was replaced with 2 ml fresh mediumwith 10% FBS; [³H]-mevalonic acid (NEN #NET 176) brought to a specificactivity of 0.8 Ci/mmol in a 1% solution of bovine serum albumin wasadded to this medium to a final concentration of 20 mM. At this time,one dish received 6 ml of a 4 mg/ml solution of jervine in ethanol(final concentration 28 mM jervine), and the other received 6 ml ofethanol. After 24 hr further incubation, cells were washed in PBS,extracted with methanol, and 1 M potassium hydroxide (KOH) added to 10%.Following a three hour incubation at 60° C., the methanol/KOH mixturewas extracted with diethyl ether, the extract dried down, resuspended inisopropanol, and subjected to reverse phase HPLC analysis by the methodof Rodriguez and Parks (Methods in Enzymology 111: 37-511985).

[0062] Treated cells synthesized reduced levels of cholesterol andaccumulated increased levels of another sterol that we haveprovisionally identified as the cholesterol precursor, zymosterol. Thenatural product jervine at these concentrations thus inhibitscholesterol biosynthesis in cultured cells in much the same manner asthe synthetic drugs discussed above, although the specific enzyme(s)affected appear to differ. Given the similarities in their teratogeniceffects, this inhibition seems likely to underlie the teratogeniceffects of both the synthetic and natural compounds.

EXAMPLES

[0063] Immunostaining and Blotting of Hh Protein

[0064] For immunostaining, stably transfected Schneider line 2 (S2)cultured cells harboring full-length Hh under metallothionein promotercontrol (Porter et al. 1995) were induced by adding CuSO4 (0.5 mM) tothe medium, incubated overnight, transferred to an 8-chamber slide(Nunc) and allowed to adhere for one hour. The cells were fixed with 4%paraformaldehyde/PBS for 10 min at room temperature (RT) and washedseveral times with PBSS (PBS containing 0.1% saponin). The cells were asfollows, several PBSS washes at every reagent change: anti-N (1:100dilution of anti Hh-N described in Lee et al 1994) for one hour,anti-rabbit-Texas Red (1:50 dilution, Jackson ImmunoResearchLaboratories) for 30 min. All incubations were performed at RT and theantibodies were diluted in 1% BSA/PBSS. The cells were mounted withVectashield (Vector Laboratories) and observed by confocal microscopy(Biorad).

[0065] For immunoblotting, medium and cells from cultures of stablytransfected S2 cells expressing full-length or truncated Hh weresuspended in SDS sample buffer, and equivalent fractions of the totalculture were loaded onto a 12% polyacrylamide gel, electrophoresed andtransferred to nitrocellulose. Amino-terminal epitopes were detectedwith anti-Hh-N antibody (Lee et al. 1994) and bound antibody wasdetected with ECL (Amersham).

[0066] Jervine Inhibition of Cholesterol Biosynthesis

[0067] Metabolic labeling and sterol analysis was essentially asdescribed (Popjak et al. 1989; Rilling et al. 1993; Rodriguez and Parks1985), with minor modifications. Briefly, COS-7 cells were plated at 35%confluence into two 60 mm dishes at 37° C. in 4 ml each of Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum(FBS). After 24 hr of growth the medium in each dish was replaced with 2ml fresh medium with 10% FBS; [3H]-mevalonic acid (NEN #NET 176) broughtto a specific activity of 0.8 Ci/mmol in a 1% solution of bovine serumalbumin was added to this medium to a final concentration of 20

M. At this time, one dish received 6

1 of a 4 mg/ml solution of jervine in ethanol (final concentration 28

M jervine), and the other received 6

1 of ethanol. After 24 hr further incubation, cells were washed in PBS,extracted with methanol, and 1 M potassium hydroxide (KOH) added to 10%.Following a three hour incubation at 60✓ C, the methanol/KOH mixture wasextracted with diethyl ether, the extract dried down, resuspended inisopropanol, and subjected to reverse phase HPLC analysis by the methodof Rodriguez and Parks (1985).

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We claim:
 1. A method for affecting cholesterol biosynthesis ortransport in a cell comprising contacting a cell with an effectiveamount of a compound that affects hedgehog, thereby affectingcholesterol biosynthesis or transport.
 2. The method of claim 1, whereinthe effect is inhibition.
 3. The method of claim 1, wherein the effectis stimulation.